Liposomal systems comprising sphingomyelin

ABSTRACT

The present disclosure provides a liposomal system comprising an aqueous medium having dispersed therein liposomes encapsulating in their intraliposomal aqueous compartment at least one active agent, the aqueous medium being in iso-osmotic equilibrium with said intraliposomal aqueous compartment, the liposomes having a membrane comprising a liposome forming lipids, at least one of which being sphingomyelin (SPM), the liposomal system having increased stability as compared to the same liposomes free of SPM, and in one embodiment being stable during long-term storage, said stability being characterized in that no more than 30% of the at least one active agent is present in the aqueous medium after said storage. Further provided by the present disclosure are a method for storage of liposomes making use of the liposomal system; use of the liposomal system for the treatment of a medical condition or for the diagnostic of a medical condition; a pharmaceutical or diagnostic composition comprising the liposomal system, and a method of treating or diagnosing of a medical condition comprising administering to a subject an amount of the liposomal system.

FIELD OF THE INVENTION

This invention relates to the field of liposome technology.

BACKGROUND OF THE INVENTION

Among other applications, liposomes are used as carriers of drugs fordelivery via a plurality of mechanisms. To this end, various types ofliposomes are used, from small unilamellar vesicles (SUV), largeunilamellar vesicles (LUV), multilamellar vesicles (MLV), multivesicularvesicles (MVV), large multivesicular vesicles (LMVV, also referred to,at times, by the term giant multivesicular vesicles, “GMV”),oligolamellar vesicles (OLV), and others. It is appreciated by thoseversed in the art that LMVV are somewhat different from unilamellarvesicles of various sizes and of the “onion like” MLV structure. In LMVVthe amount of aqueous medium forming the aqueous phase per the amount oflipid is greater than that in MLV, this potentially allowing higheramount of drug to be loaded into the aqueous phase, namely, higher drugto lipid mole ratio in the LMVV when compared to MLV system of similarsize distribution. This difference was exemplified by Grant et al. 2004[Anesthesiology 101(0:133-7, 2004] and in U.S. Pat. No. 6,162,462. Ithas been found that the difference in structure between MLV an LMVV notonly allows higher loading of the drug into the liposomes but also aprolonged release of the drug from the LMVV system.

Specifically, U.S. Pat. No. 6,162,462 discloses liposomal bupivacainecompositions in which the bupivacaine is loaded by a transmembraneammonium sulfate gradient, the liposomes being giant multivesicularvesicles (GMV, a synonym for LMVV) having a molar ratio of encapsulateddrug to lipid in said liposomal composition of at least 1.0. A specificdrug encapsulated in the liposomes of U.S. Pat. No. 6,162,462 is theamphipathic analgesic drug bupivacaine (BUP). These bupivacaine loadedLMVV have shown to be provide superior analgesia in mice and humans[Grant et al. 2004 and U.S. Pat. No. 6,162,462, ibid.]. However aphenomenon that still remains unresolved with these LMVV relates toleakage of bupivacaine from the LMVV during storage at 4° C. or roomtemperature. Thus, after time, free drug is contained in the system (theamount being above drug MTD) and the administration of the liposomalsystem containing such free drug may result in toxicity and unwantedside effects (from exposure high amounts of free drug), unfavorablepharmacokinetics and shorter duration of the therapeutic effect. Thus,there is a need in the art to provide a system where leakage of drugfrom liposomes encapsulating same during storage is reduced orprevented.

SUMMARY OF THE INVENTION

The present disclosure is based on the finding that large multivesicularvesicles (LMVV) loaded with high amount of an amphipathic drug(bupivacaine, BUP) can be stabilized, in terms of reduced BUP leakage,if the liposomes' membranes comprise sphingomyelin and the LMVV arewithin an aqueous medium being in an iso-osmotic equilibrium with theintraliposomal aqueous medium.

Thus, the present disclosure provides, in accordance with a first of itsaspects a liposomal system comprising an aqueous medium having dispersedtherein liposomes encapsulating in their intraliposomal aqueouscompartment at least one active agent, the aqueous medium being iniso-osmotic equilibrium with said intraliposomal aqueous compartment,the liposomes having a membrane comprising a liposome forming lipids, atleast one of which being sphingomyelin (SPM), the liposomal systemhaving increased stability as compared to the same liposomes free of SPM(namely, where there is no SPM in the liposome forming membrane). In oneembodiment, the liposomal system is stable during long-term storage,said stability being characterized in that no more than 30% of the atleast one active agent is present in the aqueous medium after saidstorage.

The present disclosure also provides, in accordance with a second of itsaspects, a method for storage of liposomes encapsulating in theirintraliposomal aqueous compartment at least one active agent, theliposomes having a membrane comprising liposome forming lipids, at leastone liposome forming lipid being sphingomyelin (SPM), the methodcomprising forming a liposomal system where said liposomes are dispersedin an aqueous medium being in an iso-osmotic equilibrium with theintraliposomal aqueous compartment of said liposomes and storing saidliposomal system, the liposomal system having increased stability ascompared to the same liposomes free of SPM.

Also provided by some aspects of the present disclosure is the use of aliposomal system as defined herein, for the preparation of apharmaceutical or diagnostic composition; as well as the liposomalsystem as defined for use in the treatment of a medical condition or forthe diagnostic of a medical condition.

Further, an aspect of the present disclosure provides a pharmaceuticalor diagnostic composition comprising the liposomal system as definedherein and at least one physiologically acceptable carrier.

Yet further, the present disclosure provides a method of treating ordiagnosing of a medical condition comprising administering to a subjectan amount of the liposomal system as defined herein.

In one preferred embodiment, the active agent is an amphipathiccompound, being loaded into the liposomes by remote loading technique;the SPM is synthetic or semi-synthetic C16 or C18 SPM and the liposomesare large multivesicular vesicles (LMVV).

A particular liposomal system in accordance with the present disclosurecomprises LMVV formed from a combination of at least hydrogenated soyphosphatidylcholine (HSPC), C16SPM, cholesterol and encapsulating BUP.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIGS. 1A-1B are graphs showing the release of Bupivacaine (BUP), duringstorage at 4° C. (FIG. 1A) or at 37° C. (FIG. 1B), from largemultivesicular vesicles (LMVV) of different lipid compositions (BUP tophospholipid mole ratio of each is given) which have been loaded withBUP using remote loading driven by trans-membrane ammonium sulphate (AS)gradient.

FIGS. 2A-2B are graphs showing the release of Bupivacaine (BUP), duringstorage at 4° C. (FIG. 2A) or at 37° C. (FIG. 2B), from largemultivesicular vesicles (LMVV) of different lipid compositions (BUP tophospholipid mole ratio of each is given) which have been loaded withBUP using remote loading driven by trans-membrane calcium acetate (CA)gradient.

FIGS. 3A-3B are graphs showing the release of Bupivacaine (BUP), duringstorage at 4° C. (FIG. 3A) or at 37° C. (FIG. 3B), from LMVV ofdifferent lipid compositions (HSPC/CHOL 6/4 mole ratio; HSPC/C16SPM/CHOL3/3/4 mole ratio; and HSPC100/CHOL 6/4 mole ratio, BUP to phospholipidmole ratio of each composition is given) which have been loaded with BUPusing the passive loading approach.

FIGS. 4A-4C are graphs showing the duration of analgesia in mice usingvarious liposomal systems identified in Table 8 as formulations 1 to 8(identified in the Figures with in the corresponding formulation number“x” as “lip x”), FIG. 4A showing the effect of injected volume ofliposomal BUP or in free form, the amount of BUP being constant 6mg/mouse; FIG. 4B showing the effect of 5 different LMVV formulations,the amount of BUP being constant 3 mg; and FIG. 4C which describes acomparison of the eight different LMVV formulations (Table 8) at a doseof 3 mg/mouse.

FIGS. 5A-5F are graphs comparing analgesia duration of two differentdoses of BUP (3 mg/mouse and 6 mg/mouse) for the five different LMVVformulations identified in Table 8 (“lip x” in FIGS. 5A-5E) and 2different amounts (0.375 and 0.75 mg/mouse) of non-encapsulated (free)BUP (in FIG. 5F); FIG. 5A comparing the effect of lip 2 (3 and 6 mgBUP/mouse), FIG. 5B comparing the effect of lip 3 (3 and 6 mgBUP/mouse), FIG. 5C comparing the effect of lip 4 (3 and 4.5 mgBUP/mouse), FIG. 5D comparing the effect of lip 5 (3 and 6 mg BUP), FIG.5E comparing the effect of lip 8 (3 and 6 mg BUP/mouse), and FIG. 5Fcomparing the effect of free (non liposomal) BUP at 0.375 mg/mouse usingtwo volumes (150 and 300 μl) and 0.75 mg/mouse at a volume of 150 μl.

FIG. 6 is a graph showing in vivo analgesia after 20 hours of LMVVcomprising HSPC:C16SPM:cholesterol [3/3/4] 3 mg BUP and LMVV asdescribed by Grant et al. 2004 and free BUP 0.75 mg/mouse (the maximaltolerated dose, MTD).

FIG. 7 is a graph showing in vivo analgesia after 40 hours of LMVVcomprising HSPC:C16SPM:cholesterol [3/3/4] 3 mg BUP LMVV and free BUP0.75 mg/mouse (the maximal tolerated dose, MTD).

FIGS. 8A-8E are graphs comparing the change in level of free bupivacaine(as % in storage medium) during the indicated storage period, at 4° C.of HSPC100/C16SPM/CHOL (3/3/4 mole ratio) LMVV loaded with bupivacainevia the AS trans-membrane as is when stored in various storage media(Saline, 0.5% BUP or 2.0% BUP).

DETAILED DESCRIPTION OF SOME NON-LIMITING EMBODIMENTS

The present invention is based on the understanding that existingbupivacaine liposomal formulations such as those described in U.S. Pat.No. 6,162,462, and Grant et al. (Grant et al. 2004, ibid.) have atendency to leak during long term storage at low temperatures which mayimpose a risk of toxicity when administered to subjects in need of thedrug. These bupivacaine liposomal formulations contained high drug tophospholipid ratio (>0.5 mole/mole) in large multivesicular vesicle(LMVV, referred to in U.S. Pat. No. 6,162,462 as giant multivesicularvesicles, GMV), albeit, following storage, a substantial amount of the apriori encapsulated drug was found to be present in the external medium.Thus, a novel liposomal system was designed where the amount of freebupivacaine in the medium external to the liposomes was significantlyreduced after long term storage at 4° C., as compared to the hithertoexisting bupivacaine liposomal formulations. It was further found thatwhile the liposomal system was stable during storage at 4° C., atphysiological conditions, namely, at 37° C., bupivacaine was releasedfrom the liposomes at a controlled and prolonged rate sufficient to getlong term (prolonged) analgesia.

Specifically, it has been found that liposomes comprising in theliposome's bilayer sphingomyelin at the amount of up to 75% of the totalphospholipids (or 50% of total lipids (which include ˜33 mole %cholesterol) forming the liposome's bilayer decreased the amount ofleakage without compromising the rate of bupivacaine release from theliposomes at 37° C. and without compromising the high loading of thedrug into the liposomes.

Thus, in accordance with a first of its aspects, the present disclosureprovides a liposomal system comprising an aqueous medium havingdispersed therein liposomes encapsulating in their intraliposomalaqueous compartment at least one active agent, the aqueous medium beingin iso-osmotic equilibrium with said intraliposomal aqueous compartment,the liposomes having a membrane comprising liposome forming lipids, atleast one of which being a sphingomyelin (SPM), the liposomal systembeing stable.

It has been found that the stability of the SPM containing liposomes issignificantly greater than that of liposomes which do not contain SPM intheir lipid membrane. The stability of the liposomal system is alsodetermined in terms of long-term storage, the stability beingcharacterized in that no more than 30%, at times, not more than 20% andeven not more than 10% of the at least one active agent of the system ispresent in the aqueous medium after said storage.

As used herein, the term “liposomal system” denotes a system comprisingan organized collection of lipids forming at least one type ofliposomes, and enclosing at least one intraliposomal aqueouscompartment. In addition to the liposomes, the system comprises anaqueous medium in which the liposomes are dispersed or suspended.

The aqueous medium is any water based buffer solution having a desiredosmolarity and ion concentration and is to be understood as encompassinga variety of physiologically acceptable buffers. The buffer system isgenerally a mixture of a weak acid and a soluble salt thereof, e. g.,sodium citrate/citric acid; or the monocation or dication salt of adibasic acid, e. g., potassium hydrogen tartrate; sodium hydrogentartrate, phosphoric acid/potassium dihydrogen phosphate, and phosphoricacid/disodium hydrogen phosphate. A weak acid buffer is a buffersolution with constant pH values of between 4 and 7 and a weak basebuffer is a buffer solution with constant pH values between 7 and 10.Some non-limiting examples of buffers that may be used for producing theaqueous medium in accordance with the present disclosure includephysiological saline (0.9% NaCl), phosphate buffered saline (PBS),sucrose buffer, histidine buffer etc., set at a pH of between about 4 to8, or between 5.5 to 7 (as typically used in liposomal drug deliverysystem).

In one embodiment, the aqueous medium comprises an amount of free activeagent, the presence of said free active agent in the aqueous mediumallows or participates in the formation of said iso-osmotic equilibrium.The amount of free active agent is determined such to form saidiso-osmotic equilibrium. As shown in the examples herein, the presenceof the free agent in the aqueous medium, also reduced the leakage of ethagent from the liposomes (this being comparable the same formulationwithout free drug in the aqueous medium).

In the aqueous medium are dispersed liposomes. The term “dispersed” isused to denote the distribution or suspension of the liposomes in theaqueous medium.

As appreciated, liposomes are comprises of a lipid bilayer comprisingliposome forming lipids, discussed hereinbelow, and an aqueousintraliposomal core. According to the present disclosure the aqueousmedium external to the liposomes and the intraliposomal aqueouscompartment are in iso-osmotic equilibrium. The iso-osmotic equilibriumshould be understood as meaning that the aqueous medium and the mediumof the intraliposomal aqueous compartment have similar osmolarities, thesimilarity being defined by a difference in osmolarity of not more than50 mOsmole. In accordance with one embodiment, the osmolarity of theaqueous medium and of the liposomal aqueous phase are in the range ofabout 50 to about 600 mOsm/kg, or even between about 250 to about 550mOsm/kg. The iso-osmotic equilibrium may be obtained by washing theliposomes encapsulating the active agent with the buffer solution havingan osmolarity similar to that of the intraliposomal aqueous compartment.Specifically, once the active agent is loaded into the liposomes, thenon-encapsulated agent may be washed out by the selected buffersolution.

The liposomes' membrane is a bilayer membrane and may be prepared toinclude a variety of physiologically acceptable liposome forming lipids.As used herein, the term “liposome forming lipids” is used to denoteprimarily glycerophospholipids and sphingomyelins. Theglycerophospholipids have a glycerol backbone wherein at least one,preferably two, of the hydroxyl groups at the head group is substitutedby one or two of an acyl, alkyl or alkenyl chain, a phosphate group, orcombination of any of the above, and/or derivatives of same and maycontain a chemically reactive group (such as an amine, acid, ester,aldehyde or alcohol) at the head group, thereby providing the lipid witha polar head group. The sphingomyelins consists of a ceramide unit witha phosphorylcholine moiety attached to position 1 and thus in fact is anN-acyl sphingosine The phosphocholine moiety in sphingomyelincontributes the polar head group of the sphingomyelin.

In the liposome forming lipids the acyl chain(s) are typically between14 to about 24 carbon atoms in length, and have varying degrees ofsaturation being fully, partially or non-hydrogenated lipids. Further,the lipid matrix may be of natural source, semi-synthetic or fullysynthetic lipid, and neutral, negatively or positively charged.

Examples of liposome forming glycerophospholipids include, without beinglimited thereto, glycerophospholipid. phosphatidylglycerols (PG)including dimyristoyl phosphatidylglycerol (DMPG); phosphatidylcholine(PC), including egg yolk phosphatidylcholine, dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylphosphatidyl choline(POPC), hydrogenated soy phosphatidylcholine (HSPC),distearoylphosphatidylcholine (DSPC); phosphatidic acid (PA),phosphatidylinositol (PI), phosphatidylserine (PS).

As appreciated, the liposome forming lipids may also include cationiclipids (monocationic or polycationic lipids). Cationic lipids typicallyconsist of a lipophilic moiety, such as a sterol or the same glycerolbackbone to which two acyl or two alkyl, or one acyl and one alkyl chaincontribute the hydrophobic region of the amphipathic molecule, to form alipid having an overall net positive charge. Preferably, the headgroupof the lipid carries the positive charge.

Monocationic lipids may include, for example,1,2-dimyristoyl-3-trimethylammonium propane (DMTAP)1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE); N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethyl-ammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);3β[N-(N′,N′-dimethylaminoethane) carbamoly] cholesterol (DC-Chol); anddimethyl-dioctadecylammonium (DDAB).

Polycationic lipids may include a similar lipophilic moiety as with themono cationic lipids, to which spermine or spermidine is attached. Theseinclude, without being limited thereto,N-[2-[[2,5-bis[3-aminopropyl)amino]-1-oxopentyl]amino]ethyl]-N,N-dimethyl-2,3-bis[(1-oxo-9-octadecenyl)oxy]-1-propanaminium(DOSPA), and ceramide carbamoyl spermine (CCS). The cationic lipids mayform part of a derivatized phospholipids such as the neutral lipiddioleoylphosphatidyl ethanolamine (DOPE) derivatized with polylysine toform a cationic lipopolymer.

According to the present disclosure, the liposome forming lipidcomprises at least a sphingomyelin. The term “sphingomyelin” or “SPM” asused herein denotes any N-acetyl sphingosine conjugated to aphosphocholine group, the later forming the polar head group of thesphingomyelin (N-acetylsphingosylphosphorylcholines). The acyl chainbound to the primary amino group of the sphingosine may be saturated orunsaturated, branched or unbranded. In one embodiment, the acyl chaincomprises between 12 to 24 carbon atoms (C12-C24), at times between 14to 20 carbon atoms. In some preferred embodiments, the SPM is a C16:0 orC18:0 sphingomyelin, namely, saturated C16 or C18 SPM. The SPM ispreferably a synthetic or semi-synthetic SPM, i.e. a derivative of anaturally occurring SPM and may include the natural D-erythro (2S, 3R)isomer and the non naturally occurring L-threo (2S, 3S) isomer, althoughthe former, i.e. the naturally occurring isomer is preferable.

In addition, in the context of the present disclosure, the sphingomyelinis also used to denote the corresponding dihydro species, namely, anydihydrosphingomyelins (DHSM) corresponding to the SPM defined hereinabove.

In one embodiment, the liposomal system comprises SPM content in theliposomes membrane in an amount between 25 to 75 mole % of the totalphospholipids (liposome forming lipid) in said membrane, or about 50mole % of the total lipids when including cholesterol.

In yet a further embodiment, the mole ratio between the liposome forminglipids other than SPM and said SPM is typically in the range of 1:1 to2:1, irrespective of the SPM used in accordance with the presentdisclosure.

Typically, the liposome forming lipids have when assembled into theliposome membranes have a solid ordered (SO) to liquid disordered (LD)phase transition temperature having a characteristic temperature definedas T_(m)>37° C. T_(m) is the temperature within the range of the SO toLD phase transition temperatures in which the maximal change in the heatcapacity of the phase transition occurs. Interestingly, it has beenfound and also shown hereinbelow that the combination HSPC having asolid ordered to liquid disordered with a T_(m) at ˜53° C. with C16SPMhaving its T_(m) at ˜41.4° C. surprisingly led to the formation of astable liposomal system, i.e. reduced drug leakage during 4° C. storage,as compared to a liposomal system lacking C16SPM which was less stable,namely, showing higher rate of drug leakage during 4° C. storage (i.e.same storing conditions).

The term “stablility” in the context of the present disclosure is usedto denote that the resulting liposomes were more stable (less agentbeing leaked from the liposomes during or following storage, thedifference in leakage being statistically significant) as compared tothe same liposomes, albeit free of SPM, namely, the liposome's membranedoes not comprise SPM as part of the liposome forming lipids. Thestability may also be defined that the drug loaded liposomes arechemically and physically unaltered when stored at 4° C. and for aperiod of at least 3 months. The stability is determined, for example,by measuring the amount of free active agent that present or wasreleased (leaked) to the extra-liposome aqueous medium, i.e.non-encapsulated active agent, the amount indicative of stability beingless than 30%, 20% and at times even less than 10% from the total amountof active agent in the liposomal system (the total amount includingencapsulated and non-encapsulated agent). Surprisingly, the resultspresented herein show that when comparing a liposome formulation e.g.comprising HSPC and Cholesterol with the amount of leakage of anencapsulated agent from the same formulation, albeit with SPM in thelipid membrane, leakage of the agent was reduced.

The liposomes may also comprise other lipids typically used in theformation of liposomes, e.g. for stabilization, for affecting surfacecharge, membrane fluidity and/or assist in the loading of the activeagents into the liposomes. Examples of such lipids, may include sterolssuch as cholesterol, cholesteryl hemisuccinate, cholesteryl sulfate, orany other derivatives of cholesterol.

The liposomes may further comprise lipopolymers. The term “lipopolymer”is used herein to denote a lipid substance modified at its polarheadgroup with a hydrophilic polymer. The polymer headgroup of alipopolymer is typically water-soluble and may be covalently ornon-covalently attached to a hydrophobic lipid region. Typically, thehydrophilic polymer has a molecular weight equal or above 750 Da and maybe polar or apolar. Lipopolymers such as those that may be employedaccording to the present disclosure are known to be effective forforming long-circulating liposomes. There are numerous polymers whichmay be attached to lipids to form such lipopolymers, such as, withoutbeing limited thereto, polyethylene glycol (PEG), polysialic acid,polylactic (also termed polylactide), polyglycolic acid (also termedpolyglycolide), apolylactic-polyglycolic acid, polyvinyl alcohol,polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline,polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyaspartamide,polyhydroxypropyl methacrylamide, polymethacrylamide,polydimethylacrylamide, polyvinylmethylether, polyhydroxyethyl acrylate,derivatized celluloses such as hydroxymethylcellulose orhydroxyethylcellulose. The polymers may be employed as homopolymers oras block or random copolymers. The lipids derivatized into lipopolymersmay be neutral, negatively charged, as well as positively charged. Themost commonly used and commercially available lipids derivatized intolipopolymers are those based on phosphatidyl ethanolamine (PE), usually,distearylphosphatidylethanolamine (DSPE).

One particular family of lipopolymers that may be employed according tothe present disclosure are the monomethylated PEG attached to DSPE (withdifferent lengths of PEG chains, in which the PEG polymer is linked tothe lipid via a carbamate linkage resulting in a negatively chargedlipopolymer, or the neutral methyl polyethyleneglycol distearoylglycerol(mPEG-DSG) and the neutral methyl poly ethyleneglycoloxycarbonyl-3-amino-1,2-propanediol distearoylester (mPEG-DS) [GarbuzenkoO. et al., Langmuir. 21:2560-2568 (2005)]. Another lipopolymer is thephosphatidic acid PEG (PA-PEG).

The PEG moiety has a molecular weight of the head group is from about750 Da to about 20,000 Da, at times, from about 750 Da to about 12,000Da and typically between about 1,000 Da to about 5,000 Da. One specificPEG-DSPE commonly employed in liposomes is that wherein PEG has amolecular weight of 2000 Da, designated herein ²⁰⁰⁰PEG-DSPE or^(2k)PEG-DSPE.

The liposomes of the liposomal system encapsulate at least one activeagent. Encapsulation includes the entrapment/enclosure, in theintraliposomal phase, of at least one active agent. The entrapment is anon-covalent entrapment, namely in the liposomal aqueous phase theactive agent is freely dispersed and may, under appropriate conditions,be released from the liposomes in a controlled manner.

The active agent may be a small molecular weight compound as well as apolymer (e.g. peptide, protein, nucleic acid sequence etc.). The term“active agent” is used to denote that the encapsulated agent, onceadministered has a beneficial effect, e.g. as a therapeutic, as acontrasting agent (e.g. radionuclei dyes or dye-conjugates to carrier,chromophor or fluorophor producing agent etc.), as a nutraceuticalcompound etc. The active agent may be a water soluble, hydrophiliccompound as well as an amphipathic compound.

In one embodiment, the active agent is an amphipathic compound. The term“amphipathic compound” is used to denote a active agent possessing bothhydrophilic and lipophilic properties. There are various biologicallyactive amphipathic compounds known in the art. One example includes theanti cancer compound doxorubicin. The loading of doxorubicin (e.g.,DOXIL™) into preformed liposomes is driven by transmembrane ammoniumsulfate gradient (U.S. Pat. No. 5,192,549, U.S. Pat. No. 5,316,771 andHaran et al., [Haran G, et al. (1993) Transmembrane ammonium sulfategradients in liposomes produce efficient and stable entrapment ofamphipathic weak bases. Biochim Biophys Acta. 1151(2):201-15].

In one other embodiment, the amphipathic active agent is an analgesicdrug. The analgesic drug would typically be for local analgesic. Anon-limiting group of analgesic drugs are selected from the groupconsisting of benzocaine, chloroprocaine, cocaine, cyclomethycaine,dimethocaine, propoxycaine, procaine, proparacaine, tetracaine,articaine, bupivacaine, carticaine, cinchocaine, etidocaine,levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine,ropivacaine, trimecaine, saxitoxin and tetrodotoxin. A preferred groupof analgesic drugs include, without being limited thereof, bupivacaine,lidocaine, ropivacaine, levobupivacaine, procaine, chloroprocaine,benzocaine, etidocaine, mepivacaine, prilocaine, ciprocaine, tetracaine,dibucaine, heptacaine, mesocaine, propanocaine, carbisocaine, andbutacaine. A specific analgesic drug according to the present disclosureis bupivacaine (hereinafter referred to, at times, as “BUP”).

In another embodiment, the active agent is a water soluble molecule suchas a peptide, protein or nucleic acid sequences, including, for example,cytokines, antibodies, immunostimulating oligonucleotides (ISS-ODN),siRNA etc.

As appreciated, liposomes in general may have various shapes and sizes.The liposomes may be multilamellar liposomes (MLV) or multivesiclularvesicles (MVV). MVV liposomes are known to have the form of numerousnon-concentric, closely packed internal aqueous chambers separated by anetwork of lipid membranes and enclosed in a lipid membrane. In thecontext of the present invention, the MVV are referred to as largemultivesicular vesicles (LMVV), also known in the art by the term giantmultivesicular vesicles (GMV). In accordance with one embodiment, theliposomes typically have a diameter of at least 200 nm, typically in therange of about 200 nm and 25 μm, at times between about 250 nm and 25μm.

When the liposomes are MVV or LMVV, it is to be understood that theloading of the agent into the LMVV includes containment of the agent inmore than one aqueous compartment formed by the lipid membranes, andtypically also in the aqueous environment surrounding the non-concentriclipid membrane. At times, the agent may be entrapped (embedded) in thelipid membrane, e.g. when the active agent is lipophilic compound.

The liposomal system disclosed herein is characterized by a high activeagent to lipid ratio, namely, high level of active agent per liposome.Although not exclusively, the high loading would typically depend on thetype of liposomes used, their size, the loading conditions etc. In oneembodiment, a high loading is achieved by active loading (see below) ofthe active agent into LMVV under condition of high initial active agentconcentration. In the context of the present disclosure, high loading isused to denote a loading with a active agent to lipid ratio in theresulting liposomal system of at least about 0.5 mole drug per moleliposome forming phospholipid ratio (mole/mole) (this beingcharacteristic of the LMVV according to the present disclosure).

Loading of the active agent into the liposomes may be by any techniqueknown in the art. Such techniques typically include passive loading oractive (“remote loading”) loading of agents into the liposomes.

Passive loading techniques of encapsulating agents into liposomestypically involve loading of the agent during preparation of theliposomes, e.g. by hydrating dry liposome forming lipids with a solutionof the active agent. By passive loading the agent may be associated tothe liposomal membrane or encapsulated within the aqueous core. Onemethod for passive loading was described by Bangham, et al., [Bangham AD, Standish M M, Watkins J C (1965) Diffusion of univalent ions acrossthe lamellae of swollen phospholipids. J MoI Biol. 13(1):238-52], wherean aqueous phase containing the agent of interest is put into contactwith a film of dried liposomes-forming lipids deposited on the walls ofa reaction vessel. Upon agitation by mechanical means, swelling of thelipids occurs and multilamellar vesicles (MLV) are thus formed. Afurther method for passive loading is the Reverse Phase Evaporation(REV) method described by Szoka and Papahadjopoulos, [Szoka F. C. Jr₅Papahadjopoulos D. (1978) Procedure for preparation of liposomes withlarge internal aqueous space and high capture by reverse-phaseevaporation. Proc Natl Acad Sci USA. 75(9):4194-8.], according to whicha solution of lipids in a water insoluble organic solvent is emulsifiedin an aqueous carrier phase and the organic solvent is subsequentlyremoved under reduced pressure. Other methods of passive loading includesubjecting liposomes to successive dehydration and rehydrationtreatment, or freezing and thawing. Dehydration is carried out byevaporation or freeze-drying [Kirby C and Gregoriadis G (1984)Dehydration-Rehydration Vesicles: A Simple Method for High Yield DrugEntrapment in Liposomes. Nat. Biotechnol. 2, 979-984], or mixingliposomes prepared by sonication in aqueous solution with the solute tobe encapsulated, and the mixture is dried under nitrogen in a rotatingflask. Upon rehydration, large liposomes are produced in which asignificant fraction of the solute has been encapsulated [Shew R L,Deamer D W. (1985) A novel method for encapsulation of macromolecules inliposomes. Biochim Biophys Acta. 816(1):1-8]. Loading may be improvedco-lyophilizing the active agent with the dried liposome forming lipids[International Patent Application Publication No. WO03000227]

Active loading techniques are also used. For example, liposomes may beloaded using an ion gradient or pH gradient as the pre-formed liposomeloading driving force. Loading using a pH gradient may be carried outaccording to methods described in U.S. Pat. Nos. 5,616,341, 5,736,155and 5,785,987, U.S. Pat. No. 5,192,549, U.S. Pat. No. 5,316,771 andHaran et al., [Haran G, et al. (1993) Transmembrane ammonium sulfategradients in liposomes produce efficient and stable entrapment ofamphipathic weak bases. Biochim Biophys Acta. 1151(2):201-15],incorporated herein by reference. The pH gradient may be calciumcitrate-based or ammonium sulphate-based gradient.

According to one embodiment, the liposomes have the form ofmultilamellar vesicles (MLV) or multivesicular vesicles (MVV),preferably, large multivesicular vesicles (LMVV).

The present disclosure also provides a method for storage of liposomesas defined above, i.e. encapsulating in their intraliposomal aqueouscompartment at least one active agent, the liposomes having a membranecomprising liposome forming lipids, at least one liposome forming lipidbeing sphingomyelin (SPM), the method comprising forming a liposomalsystem where said liposomes are dispersed in an aqueous medium being inan iso-osmotic equilibrium with the intraliposomal aqueous compartmentof said liposomes and storing said liposomal system, whereby no morethan 30%, at times no more than 20% and even no more than 10% of the atleast one active agent is present in the aqueous medium after saidstorage.

The method allows long term stable storage (at low temperatures, e.g. 4°C.) of the liposomes. While at minimum stable storage is for a period of3 months, as will be shown in the following non-limiting examples,stable storage was also obtained for a period of four months (120 days),4.5 months and even up to 6 months storing at 4° C. However, asindicated above, the stability would be retained at any othertemperature that is lower than the physiological temperature of thebody, namely, below 37° C. When referring to lower temperatures it is tobe understood that the reasonable storage temperature should be at least15° C. below body core temperature, i.e. below 22° C. According to oneembodiment, storing is at a temperature between about 2° C. to 8° C.

Due to the low leakage of the active agent during storage ofSPM-containing LMVV there it has been found that there is no need towash the liposomal system prior to administration to a subject in needthereof. The liposomal system may be administered to the subject in needthereof as is or may be combined with a physiologically acceptableadditive.

Thus, the present invention also provides the use of the liposomalsystem as defined hereinabove for the preparation of a pharmaceutical ordiagnostic composition, for, respectively, treatment of a medicalcondition or for diagnostic purposes. The composition typicallycomprises, in addition to said liposomal system, at least onephysiologically acceptable additive.

Further, the present invention provides a method for the treatment ordiagnostic of a medical condition, the method comprising administeringto a subject in need of said treatment or diagnostic an amount of theliposomal system as defined hereinabove or physiologically acceptablecomposition comprising the same.

The liposomal system alone or in combination with physiologicallyacceptable additives may be administered by any route acceptable in theart. According to one embodiment, the administration of the liposomalsystem is by parenteral injection or infusion. This would include,without being limited thereto, intravenous, intraarterial,intramuscular, intracerebral, intracerebroventricular, intracardiac,subcutaneous, intraosseous (into the bone marrow), intradermal,intratheacal, intraperitoneal, intravesical, and intracavernosal andepiduaral (peridural) injection or infusion. Pareneral administrationmay also include transdermal, e.g. by transdermal patches, transmucosal(e.g. by diffusion or injection into the peritoneum), inhalation andintravitreal (through the eye).

When the active agent is an analgesic drug, a preferred mode ofadministration is local administration by any acceptable route, as canbe determined by a medical doctor or any other appropriate physician.

The amount of liposomal system administered, and thereby the amount ofactive agent encapsulated therein should be effective to achieve thedesired effect by the active agent, at the target site. For example, ifthe active agent is a drug, the amount of the liposomal systems shouldbe determined so that at the target site the amount of the drugencapsulated therein is sufficient to achieve the desired therapeuticeffect. Such desired therapeutic effect may include, without beinglimited thereto, amelioration of symptoms associated with a medicalcondition, prevention of the manifestation of symptoms associated with amedical condition, slow down of a progression state of a medicalcondition, enhance of onset of a remission period, prevent or slow downirreversible damage caused by the medical condition, lessen the severityof the medical condition, cure the medical condition or prevent it fromdeveloping, etc. The medical condition to be treated by the liposomalsystem may be any such condition treatable by the active agentencapsulated in the liposomes according to the present disclosure.

Further, if the active agent may be a diagnostic agent. To this end, theamount of the liposomal system should be such that it would be possibleto image the marker at the target site.

The amount of the liposomal systems will be determined by suchconsiderations as may be known in the art, typically using appropriatelydesigned clinical trials (dose range studies etc.).

As used herein, the forms “a”, “an” and “the” include singular as wellas plural references unless the context clearly dictates otherwise. Forexample, the term “a liposome forming lipid” includes one or more lipidscapable of forming a liposome.

Further, as used herein, the term “comprising” is intended to mean thatthe liposomal system include the recited constituents, i.e. the liposomeforming lipid, SPM and the active agent, but not excluding otherelements, such as physiologically acceptable carriers and excipients aswell as other active agents. The term “consisting essentially of” isused to define liposomal systems which include the recited elements butexclude other elements that may have an essential significance on theeffect to be achieved by the liposomal system. “Consisting of” shallthus mean excluding more than trace amounts of other elements.Embodiments defined by each of these transition terms are within thescope of this invention.

Further, all numerical values, e.g. when referring the amounts or rangesof the elements constituting the liposomal system comprising theelements recited, are approximations which are varied (+) or (−) by upto 20%, at times by up to 10% of from the stated values. It is to beunderstood, even if not always explicitly stated that all numericaldesignations are preceded by the term “about”.

The invention will now be exemplified in the following description ofexperiments that were carried out in accordance with the invention. Itis to be understood that these examples are intended to be in the natureof illustration rather than of limitation. Obviously, many modificationsand variations of these examples are possible in light of the aboveteaching. It is therefore, to be understood that within the scope of theappended claims, the invention may be practiced otherwise, in a myriadof possible ways, than as specifically described hereinbelow.

DESCRIPTION OF SOME NON-LIMITING EXAMPLE Materials

Drugs:

Bupivacaine hydrochloride (B UP) USP XXIII (Orgamol, SA, Evionnaz,Switzerland).

Methylprednisolone sodium succinate (MPS) (PHARMACIA NV/SAPuurs-Belgium).

Lipids:

Cholesterol (CHOL) (NF; Solvay Pharmaceuticals (Veenedaal, Netherlands).

Fully hydrogenated soy phosphatidylcholine (HSPC-100), Phospholipon®100H batch no 50190 (Phospholipids GmbH Nattermannallee 1*D 50829 Koln,Germany). HSPC100 is 99.5 pure, i.e. comprising lysoPC and fatty acid inan amount less than the detectable limit.

Fully hydrogenated soy phosphatidylcholine (HSPC) (Lipoid Gmbh,Ludwigshafen, Germany). 98.0 pure, i.e. comprising less than 1.2% lysoPCand about 1% fatty acid.

Fully synthetic N-Palmitoyl-D-erythro-sphingosine-1-phosphocholine,N-palmitoyl sphingomyelin, (C16-SPM) >98% pure, Lot no. 546701 (BiolabLtd., POB 34038 Jerusalem 91340).

Buffer:

Ammonium sulfate (AS, MERCK);

Calcium acetate monohydrate (CA, Aldrich);

Calcium chloride-dihydrate (MERCK);

Methods Preparation of Drug Loaded LMVV Preparation of Large MultiVesicular Vesicles (LMVV)

Powder mixtures of lipids at the desired mole ratio (see Table 1 fordetails regarding constituents and mole ratios) were dissolved inethanol at 60-65° C. and added to an aqueous solution (ammonium sulfate(AS), calcium acetate (CA) or another buffer, as indicated below) toreach a final phospholipid (PL) concentration of 60 mM and final ethanolconcentration of 10%.

The resulting solutions were mixed for 30 min at 65° C. to obtainmultilamellar vesicles (MLV). Alternative methods to prepare MLV canalso be used (see for example: Barenholz & Crommelin, 1994, In:Encyclopedia of Pharmaceutical Technology. (Swarbrick, J. and Boylan, J.C., Eds.), Vol. 9, Marcel Dekker, NY pp. 1-39).

LMVV were prepared from the MLV with the desired aqueous phase (forexample: ammonium sulfate 250 mM or 127 mM, calcium acetate 250 mM, or200 mM; or a desired buffer) from the MLV by exposing the MLV to 10cycles of freezing in liquid nitrogen and thawing in a water bath at 60°C. thereby forming the LMVV. At each cycle, each 1 ml of dispersed LMVVsolution was kept at the liquid nitrogen for 1 minute. For example, adispersion of 3 ml was kept in liquid nitrogen for 3 minutes.

Gradient Creation

Transmembrane AS or CA gradient were created by removal of AS or CA(respectively) from the extra liposome aqueous phase and replacing itwith NaCl.

Three methods were used for creating the pH gradient:

(i) Centrifugation (Grant et al 2004, ibid.) for both AS and CAgradients at 1000 g, for 5 min and temperature of 4° C. Supernatant wasremoved and pellet was washed with saline at 4° C. The washing processwas repeated 7 times.

(ii) Dialysis using MWCO 12-14000 Dalton dialysis tubing

(iii) Diafiltrating using Midjet benchtop system with hollow fibercartridge 500000 NMWC (GE Healthcare Bio-Sciences Corp. Westborough,Mass. 01581 USA).

Loading of Bupivacaine

LMVV were loaded with Bupivacaine (B UP) using two alternativeapproaches:

Remote loading of preformed liposomes having a trans-membrane ammoniumsulfate (AS) gradient (Haran et al., (1993), BBA, 1151 201-215),modified to fit the LMVV (Grant et al 2004, ibid.); or into preformedLMVV having a trans-membrane calcium acetate (CA) gradient (Clerc &Barenholz. (1995), BBA, 1240, 65-257, Avnir et al (2008) Arthritis &Rheumatism, 58, 119-129). This method makes use of the fact that BUP,like doxorubicin, is an amphipathic weak base.

(ii) Passive loading was performed by lipid hydration using aqueoussolutions of BUP to form the BUP loaded MLV from which BUP loaded LMVVwere prepared as described above (LMVV preparation).

In both approaches loading was performed at 60-65° C., which is abovethe HSPC and C16SPM solid-ordered (SO) to liquid-disordered (LD) phasetransition temperature range (T_(m)). It is noted that HSPC and C16SPMare the liposome-forming lipids of the LMVV described here.

For remote loading, loading was performed for 30 min. at 60-65° C. using4.5%, 5.5%, or 5.7% BUP, which is equivalent to osmolarity of(saline=0.9% weight per volume), or 6% BUP in distilled water as theliposome external aqueous phase. An amount 0.5 ml of a wet LMVV pelletand 2 ml of BUP solution were used for the remote loading. The mixturewas then cooled to 4° C. overnight.

Passive loading of BUP was performed by hydrating the ethanol lipidsolution with aqueous solution of distilled water containing 4.5% (231mOsm/kg), or 5.5% (285 mOsm/kg), or 6% (301 mOsm/kg) or 7% (346mOsm/kg), or 8% (373 mOsm/kg) or 10% (454 mOsm/kg) BUP (W/V) at 65° C.for 30 min. For this process 0.5 ml ethanolic lipids solution containing225 mg phospholipids and 77 mg CHOL were used. This solution was mixedwith 5 ml of one of the above indicated BUP aqueous solutions. Thesuspension was processed by 10 freezing and thawing cycles (as describedabove) and than kept overnight in a cold room (4-6° C.).

Free Drug Removal

Non-encapsulated BUP was removed from LMVV by washing with saline (1 mlliposomes/4 ml saline) and centrifuging the dispersion at 1000 g for 5min at 4-5° C. The washing process was repeated 7 times. The finalmedium (referred to herein as the “aqueous medium”) used to replaceextra-liposome from CA gradient loaded liposomes was PBS. The use of PBSwas preferred over saline. AS and the medium used for passive loading ofliposomes was replaced and LMVV were washed with un-buffered saline.

The LMVV was concentrated to a final solution of 2% BUP for the passiveloading and AS gradient loading. For CA gradient loading LMVV with 1%BUP final concentration was used, due to the large volume of these LMVV.These concentrations were close to the highest concentrations used forinjection of BUP.

The stability of LMVV thus formed was measured with respect to therelease rate of BUP from liposomes during storage at 4° C.

Bupivacaine Loading Under Iso-Osmotic Conditions

When referring to iso-osmotic conditions, it should be understood tomean that the osmolarity of the intraliposomal aqueous core an theexternal medium inside and outside the liposomes are essentiallyidentical or close, all as defined hereinabove.

Three osmomolar concentrations were tested:

(i) 280 mOsm/kg isoosmotic to physiological saline (0.9% NaCl)condition: the AS and CA gradient LMVV were prepared with ˜20 mg/ml ASor CA solution adjusted by AS or CA solutions to 280 mOsm/kg. BUPloading concentration was 5.7% BUP in water or 4.5% BUP in NaCl solutionto reach 280 mOsm/kg.

(ii) 550 mOsm/kg, isoosmotic to 250 mM AS: the washing solution forcreating the AS gradient and the solution for removal of the free drugafter loading was NaCl solution. adjusted to 550 mOsm/kg. The drugloading conc. was 4.5% BUP in NaCl solution, or 4.5% BUP in sucrose sol.to make 550 mOsm/kg.

(iii) 650 mOs, iso-osmotic to 250 mM CA.

Bupivacaine to Lipids Ratio

BUP was loaded into AS-LMVV using three types of BUP to lipid v/vratios:

(i) wet LMVV pellet: 5.7% BUP:lipid, 1:4 vol/vol.

(ii) wet LMVV pellet: 5.7% BUP:lipid, 1:2 vol/vol.

(iii) wet LMVV pellet: 5.7% BUP:lipid 1:1 vol/vol.

The characteristics of the resulting LMVV are provided in Table 1:

TABLE 1 BUP loaded LMVV Lipid/Chol ratio Loading method Mean size (μm)SPM/CHOL 6/4 CA gradient 8.33 ± 4.71 SPM/CHOL 6/4 AS gradient 5.7 ± 2.6HSPC/CHOL 6/4 passive 6.0 ± 3.2

Further, FIGS. 1A and 1B compare the loading stabilities of BUP-LMVV(prepared by similar procedure, albeit with H100), as measured withrespect to release rate at 4° C. (FIG. 1A) and 37° C. (FIG. 1B). Thecomparison relates to different lipid compositions of LMVV as follows:

-   -   (i) Previous formulation of HSPC (of Lipoid GmbH) and CHOL as        described in U.S. Pat. No. 6,162,46, the content of which is        incorporated herein by reference;    -   (ii) HSPC-100 (Phospholipids GmbH, Germany) and CHOL;    -   (iii) HSPC/C16SPM and CHOL;    -   (iv) HSPC 100/ C16SPM and CHOL.

The data presented in FIGS. 1A and 1B show that the release rates of BUPduring 60 days storage at 4° C. of the HSPC/CHOL liposomes was thehighest, followed by the release rate from HSPC100/CHOL liposomes. Thelowest release rate was achieved for HSPC100/C16SPM/CHOL liposomes. In24 hours, the release at 37° C. reaches the level of 60% to 70% of theBUP from the liposome—this being without reaching a plateau. It was thusconcluded that although a slight lower loading of BUP (lower BUP/PLratio) reached with the LMVV composed of HSPC100/C16SPM/CHOL, the lowrelease rate of BUP from this particular formulation at 4° C. renderedthis combination a preferred formulation. It was thus further concludedthat the presence of SPM reduced leakage as compared to the sameformulation without SPM.

The release rate from liposomes comprising HSPC100/C16SPM/CHOL 3/3/4(either SUV or LMVV as indicated) employing the different loadingtechniques, different active agents (BUP or MPS, the “Drug”) anddifferent aqueous medium (washing buffer) were examined. The results arepresented in Table 2.

TABLE 2 Drug to lipid ratio and stability loading (at 4° C.) ofliposomes formed from HSPC100/C16SPM/CHOL 3/3/4 Aqueous Drug/PL % Drugrelease at 4° C. Liposome type Loading technique medium mole ratio 17 d21 d 35 d 40 d 76 d 90 d 120 d 4.5 month 6 month LMVV-BUP Passive by4.5% BUP Saline2% BUP 1.5 17.8 LMVV-BUP Passive by 5.5% BUP Saline2% BUP1.7 20.9 36.3 LMVV-BUP Passive by 6% BUP Saline2% BUP 1.7 23.5 36LMVV-BUP Passive by 7% BUP Saline2% BUP 1.9 25.8 40.6 LMVV-BUP 250 mM CAgradient PBS, 1% BUP 0.8 11 19.9 44 LMVV-BUP 107 mm CA gradientSaline0.6% BUP 1.2 9 36.2 LMVV-BUP 107 mm CA gradient Saline0.7% BUP 1.17.5 43 LMVV-BUP 250 mm AS gradient Saline 2% BUP 1.6 8 11.1 21 LMVV-BUP250 mm AS gradient 1.75% NaCl 1.4 2.5 9.9 22 LMVV-BUP 250 MM AS gradient1.75% NaCl 2 8 9 LMVV-BUP 127 mm AS gradient Saline0.9% BUP 2.3 3 9.6 13LMVV-BUP 127 mm AS gradient Saline0.7% BUP 1.5 2.8 13.5 LMVV-BUP 127 mmAS gradient saline 1.5 3.3 20 LMVV-MPS 107 mm CA gradient saline 0.6 1.4SUV-MPS 250 mm CA gradient saline 0.3 20 ml LMVV- 127 mm AS gradientsaline 1.35 5 9 11.1 BUP 20 ml LMVV- 127 mm AS gradient saline 1.56 3.3BUP dialysis tube 10 ml LMVV- 127 mm AS gradient saline 1.17 BUPdiafiltration

FIGS. 2A and 2B demonstrate the release rate at 4° C. (FIG. 2A) and 37°C. (FIG. 2B) of BUP from LMVV having the same lipid compositions as usedin FIGS. 1A-1B, wherein BUP was remotely loaded using Ca acetategradient. The SPM used was C16 SPM, and comparison with HSPC/SPM/CHOLand HSPC100/SPM/CHOL was also made at 4° C.

The ratio BUP/PL for the CA gradient loading was lower than thatobtained for the AS gradient loading. Stability was assessed from therelease at 4° C. This ratio was also lower (i.e. higher release rate)than that obtained for LMVV remote loaded by AS gradient at 37° C. Therelease rates are similar to those of the LMVV loaded BUP by ASgradient, except that rate of release is faster at the first 10 hoursfollowed by an almost plateau. It is apparent from FIG. 2A that theHSPC100 LMVV has better stability (i.e. lower leakage at 4° C.) thanHSPC based LMVV, and that C16 SPM effect on improving stability is muchgreater than the difference between the two HSPC preparations. C 16 SPMalso reduces leakage rate for both HSPC compositions by a similarextent.

FIGS. 3A and 3B demonstrate the release rate at 4° C. (FIG. 3A) and 37°C. (FIG. 3B) of BUP loaded LMVV of the same lipid compositions used inFIGS. 1A and 1B, wherein LMVV were passively loaded with BUP. The SPMused is C16 SPM, and a comparison of HSPC/SPM/CHOL and HSPC100/SPM/CHOLwas also made at 4° C.

In general, release rates at 4° C., for passively loaded LMVV of the 3lipid compositions used, were higher than for the remote loading via CAgradient and even higher when compared with AS remote loading LMVV.

However the effect of LMVV lipid composition on release rates at 4° C.and 37° C. were similar (but larger in magnitude) to that observed forthe remote loading driven by AS and CA gradient, thus indicating thatthe ion gradient driven remote loading increases loading stability at 4°C.

LMVV Optimization

Various formulations with different mole ratio of HSPC100:C16SPM wereprepared in order to determined the optimized ratio between these twoconstituents. The different formulations are provided in Tables 3A and3B.

TABLE 3A Effect of HSPC100:C16SPM mole ratio in HSPC100/C16SPM/CHOL LMVVformed by active loading with AS gradient % SPM/ BUP HSPC100 BUP/PL %BUP release at 4° C. load- mole mole 2 3.5 ing ratio ratio 8 d 22 d 30 d38 d month month 4.5 0/1 2.2 2.5 8.2 18.9 4.5 1/0 1.8 4 9.5 15.5 4.5 1/11.68 8 5.7 1/1 1.96 7.5 8.7 5.7 5/4 2.03 5.2 7 5.7 2/1 1.5 5.8 7.8 5.77/2 1.6 5.3 7.5 5.7 0/1 1.8 4.3 5.7 1/1 1.55 2.6 5.7 2/1 1.44 2.4

TABLE 3B Effect of HSPC100:C16SPM mole ratio in HSPC100/C16SPM/CHOL LMVVformed by active loading with CA gradient. % SPM/ BUP HSPC100 BUP/PL %BUP release at 4° C. load- mole mole 2 3.5 ing ratio ratio 8 d 22 d 30 d38 d month month 4.5 0/1  1.7 2 19.2 41.2 4.5 1/0  1.45 7.4 8.8 20.8 4.51/1  1.77 15 4.5 0/1* 1.16 2 25.8 4.5 1/1* 1.5 3 12.6 34 4.5 1/3* 1.53.7 16 41 *HSPC and not HSPC100

Further, pre-formed LMVV were centrifuged for 5 min at 4° C. at 2000 gto give packed LMVV. For remote loading the packed LMVV were suspendedin various volumes of 5.7% BUP. The volume ratio of BUP to LMVV or PL isgiven in Table 4.

TABLE 4 Optimization of passive loading to the volume ratio of 5.7% BUPto packed LMW (during loading). BUP/LMVV % free BUP volume ratio* BUP/PLmole ratio t = 0 4 1.17 0.4 2 1.23 0.6 1 1.13 2.8

In Vivo Experiments Bupivacaine Loaded LMVV Preparations:

Eight formulations were prepared (as specified below) under sterileconditions and were tested for sterility in the Clinical MicrobiologyDepartment, Hadassah Hospital, Jerusalem, Israel. The liposomes wereshipped from Jerusalem Israel to Dr G. J. Grant, Department ofAnesthesiology, NYU, School of Medicine, NYC, USA at controlledtemperature of 2° C.-8° C. HPLC analysis (not shown) before shipment andafter arrival to destination indicated that no leakage during shipmenttook place.

TABLE 7 Liposome Composition Characterization Ratio Pi Bupivicaine/Pellet Total % of Bupivicaine μmol/ml = Pi mM date of sample Liposomesvolume volume free (total) mmol/1 = Bupivicane/ Sample preparationnumber Gradient sort type ml bupiv. bupiv. mM mM mM Pi H100/SPM_(c16)/15 Jul. 2007 1 AS (in saline) MLV 3.5 15 5.08 17.11 28.12 0.61 CHOL3/3/4 1 ml lipos (instead 0.5 ml) + 2 ml 4.5% bup. 09 Jul. 2007 2 CaAcMLV 4 15 3.09 17.86 19.53 0.91 (in PBS) 10 Jul. 2007 3 AS LMVV 5 15 2.8127.36 13.71 2.00 (in saline) 11 Jul. 2007 & 4 CaAc LMVV 15 30 3.56 17.2821.06 0.82 15 Jul. 2007 (in PBS) H100/CHOL 16 Jul. 2007 5 AS LMVV 7 153.27 32.23 15.91 2.03 6/4 (in saline) HS 16 Jul. 2007 6 AS LMVV 6 156.81 33.32 14.89 2.24 (in saline) H100/CHOL 17 Jul. 2007 7 CaAc LMVV 715 6.11 14.67 19.82 0.74 6/4 (in PBS) H100/SPM_(c16)/ 18 Jul. 2007 8 6%passive LMVV 4 15 1.20 23.00 20.13 1.14 CHOL 3/3/4

All liposomal formulations were analyzed for free BUP and total BUPbefore the in vivo experiment and concentrated to reach the level of 2%(w/w) BUP (liposomes formulations #1, 2, 3, 5, 6, 8) or 1% (w/w) BUP(liposomes formulations #4, 7). BUP was loaded into the liposomes eitherby active loading (CA or AS gradient) or by passive loading.

TABLE 8 Liposome composition analysis prior to in vivo experimentationLiposome # Lipids* Loading technique type % free BUP 1 H100/SPM/CHOL ASgradient MLV 3.88 2 H100/SPM/CHOL CA gradient MLV 3.95 3 H100/SPM/CHOLAS gradient LMVV 3.69 4 H100/SPM/CHOL CA gradient LMVV 4.52 5 H100/CHOLAS gradient LMVV 3.68 6 HSPC/CHOL AS gradient LMVV 7.80 7 H100/CHOL CAgradient LMVV 7.66 8 H100/SPM/CHOL 6% BUP passive LMVV 1.90 loading*with SPM the ratio is 3/3/4 and without SPM the ratio is 6/4

Analgesic Efficacy in Mouse Model:

Testing for analgesia was done by electrical stimulation of the skindirectly overlying the abdomen at the site of injection using a currentgenerator (model S48, Grass Instruments).

Mice (male Swiss-Webster, 26±3 gr) were tested prior to injection todetermine the vocalization threshold than were injected withencapsulation BUP liposomes than determine analgesic duration (G. J.Grant et al, pharmaceutical research, vol 18, no. 3, 336-343, 2001).

The duration of the main in vivo screening study was 2 days and startedafter a preliminary study using two different injection volumes offormulation #4 (referred to as the PILOT in Table 9A) was performed.

In order to evaluate the effect of altering the volume and BUPconcentration of the injection, in each group, three mice received 150μL of the 2% formulation and 3 mice received 300 μL of a 1:1 diluted 2%formulation.

It has been previously determined (Grant et al. 2004, ibid., Bolotin etal. 2000, ibid. and U.S. Pat. No. 6,162,462) LMVV (GMV) encapsulated BUPprovide an analgesic effect for approximately 75 minutes post injection.

The analgesic efficacy of the various formulations 1 to 8, at differentBIP concentration, different injection volume etc. is presented inTables 9A to 9C. In these Tables, an numeric score of “1” denotes fullanalgesia, a numeric score of “0” was given when there was no analgesiceffect, and a numeric value of “10” when there was partial analgesia. Inthe following tables the numeric value “10” is replaced by “0.5”.

In Table 9A results of mice injected with LMVV formulation #4, two micewith 300 μl and two mice with 150 μl are presented as “PILOT 1-4”Testing was done at 4, 17, and 21 hours following injection.

FIGS. 4A-4C, 5A-5F, 6 and 7 show the duration of analgesia. Thedifference in these figures is in the formulations used, FIGS. 4 and 5making use of the various formulations identified in Table 8, and FIGS.6 and 7 making use of HSPC100/C16SPM/CHOL (3/3/4). The in vivo resultsshow that SPM containing liposomes have a significantly greateranalgesic effect as compared to free BUP. These results specificallyshow that the inclusion of SPM into the liposomes did not reduce theanalgesic effect to the system, as compared to prior art formulations[Grant et al. 2004, ibid.].

TABLE 9A Duration of analgesia at different BUP concentrations(administered as liposomal-BUP) and different injected volumes Aug. 9,2007 1 indicates mice under analgesia, 0 indicates mice lacks analgesia;10 indicates mice is under partial analgesia Note: On Aug. 8, 2007, weinjected four animals with LMW formulation #4 (2 animals with 300 ul and2 mice with 150 ul); testing was done at 4, 17, and 21 hours. These arelabeled “PILOT” in the spreadsheet below animal # lipo # bup conc volume(ul) mg Bup 4 hr 8 hr 12 hr 15 hr 18 hr 21 hr 1 1 2% 150 3 1 1 10 0 0 02 1 2% 150 3 1 1 1 1 0 0 3 1 2% 150 3 1 1 1 1 0 0 4 1 1% 300 3 1 1 1 0 00 5 1 1% 300 3 1 1 1 0 0 0 6 1 1% 300 3 1 1 1 0 0 0 7 2 2% 150 3 1 1 1 10 0 8 2 2% 150 3 1 1 0 0 0 0 9 2 2% 150 3 1 1 1 0 0 0 10 2 1% 300 3 1 11 1 0 0 11 2 1% 300 3 1 1 0 0 0 0 12 2 1% 300 3 1 1 0 0 0 0 13 3 2% 1503 1 1 1 1 0 0 14 3 2% 150 3 1 1 1 0 0 0 15 3 2% 160 3 1 1 1 1 0 0 16 31% 300 3 1 1 1 0 0 0 17 3 1% 300 3 1 1 1 1 1 0 18 3 1% 300 3 1 1 1 1 0 019 4 1% 300 3 1 1 1 1 1 0 20 4 1% 300 3 animal eliminated from study 214 1% 300 3 1 1 1 10 10 0 22 4 1% 300 3 1 1 1 1 10 10 23 4 1% 300 3 1 1 11 10 0 24 4 1% 300 3 1 1 1 1 0 0 17 hr PILOT 1 4 1% 300 3 1 1 0 PILOT 24 1% 300 3 1 1 0 PILOT 3 4 1% 150 1.5 1 0 PILOT 4 4 1% 150 1.5 1 0 25 52% 150 3 1 1 1 0 0 0 26 5 2% 150 3 1 1 1 1 1 0 27 5 2% 150 3 1 1 1 0 0 028 5 1% 300 3 1 1 1 1 0 0 29 5 1% 300 3 1 1 1 0 0 0 30 5 1% 300 3 1 1 100 0 0 31 6 2% 150 3 1 1 0 1 0 0 32 6 2% 150 3 1 1 1 1 0 0 33 6 2% 150 31 0 0 0 0 0 34 6 1% 300 3 1 1 0 0 0 0 35 6 1% 300 3 1 1 1 10 10 0 36 61% 300 3 1 1 1 0 0 0 37 7 1% 300 3 1 1 1 10 10 0 38 7 1% 300 3 1 1 1 1 00 39 7 1% 300 3 1 0 0 0 0 0 40 7 1% 300 3 1 1 0 0 0 0 41 7 1% 300 3 1 11 1 0 0 42 7 1% 300 3 1 1 1 1 0 0 43 8 2% 150 3 1 1 1 1 0 0 44 8 2% 1503 1 1 1 0 0 0 45 8 2% 150 3 1 1 1 0 0 0 46 8 1% 300 3 1 1 1 1 0 0 47 81% 300 3 1 1 1 1 0 0 48 8 1% 300 3 1 1 1 0 0 0

TABLE 9B Analgesic effect at different PBU concentrations and atdifferent injected volumes Aug. 13, 2007 Standard Bupivacaine (Control)1 = analgesia; 0 = no analgesia; 10 = partial analgesia Mouse # Bup ConcVolume(ul) mg Bup 15 min 30 min 45 min 60 min 75 min 90 min 105 min 120min 135 min 1 0.25% 150 0.375 1 1 1 0 0 0 2 0.25% 150 0.375 1 1 1 0 0 03 0.25% 150 0.375 1 1 1 0 0 0 4 0.25% 150 0.375 1 1 1 1 0 0 5 0.25% 1500.375 1 1 1 1 10 0 6 0.25% 150 0.375 1 1 1 0 0 0 7 0.25% 150 0.375 1 1 110 0 0 8 0.25% 150 0.375 1 1 1 0 0 0 1 0.25% 300 0.75 1 1 1 1 0 0 0 0 02 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 3 0.25% 300 0.75 1 1 1 1 1 10 10 0 04 0.25% 300 0.75 1 1 1 1 1 10 0 0 0 5 0.25% 300 0.75 1 1 1 1 1 1 1 10 06 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 7 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 80.25% 300 0.75 1 1 1 1 1 1 1 0 0 1 0.50% 150 0.75 1 1 1 1 1 0 0 2 0.50%150 0.75 1 1 1 1 1 0 0 3 0.50% 150 0.75 1 1 1 1 1 10 0 4 0.50% 150 0.751 1 1 1 0 0 0 5 0.50% 150 0.75 1 1 1 1 1 1 0 6 0.50% 150 0.75 1 1 1 1 110 0 7 0.50% 150 0.75 1 1 1 1 1 1 0 8 0.50% 150 0.75 1 1 1 10 10 0 0Liposomal (LMVV) Bupivacaine Pilot Study Mouse # LipoForm# Conc. Volumemg Bup 15 hr 18 hr 21 hr 1 3 2% 300 6 1 1 10 2 3 2% 300 6 1 1 0 1 4 1%450 4.5 1 1 0 2 4 1% 450 4.5 1 1 0 1 5 2% 300 6 1 1 0 2 5 2% 300 6 0 1 0

TABLE 9C Analgesic effect at different PBU concentrations and differentinjected volumes Aug. 13, 2007 Standard Bupivacaine (Control) 1 =analgesia; 0 = no analgesia; 10 = partial analgesia Mouse Bup Volume 1530 45 60 75 90 105 120 135 # Conc (ul) mg Bup min min min min min minmin min min 1 0.25% 150 0.375 1 1 1 0 0 0 2 0.25% 150 0.375 1 1 1 0 0 03 0.25% 150 0.375 1 1 1 0 0 0 4 0.25% 150 0.375 1 1 1 1 0 0 5 0.25% 1500.375 1 1 1 1 10 0 6 0.25% 150 0.375 1 1 1 0 0 0 7 0.25% 150 0.375 1 1 110 0 0 8 0.25% 150 0.375 1 1 1 0 0 0 1 0.25% 300 0.75 1 1 1 1 0 0 0 0 02 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 3 0.25% 300 0.75 1 1 1 1 1 10 10 0 04 0.25% 300 0.75 1 1 1 1 1 10 0 0 0 5 0.25% 300 0.75 1 1 1 1 1 1 1 10 06 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 7 0.25% 300 0.75 1 1 1 1 1 1 0 0 0 80.25% 300 0.75 1 1 1 1 1 1 1 0 0 1 0.50% 150 0.75 1 1 1 1 1 0 0 2 0.50%150 0.75 1 1 1 1 1 0 0 3 0.50% 150 0.75 1 1 1 1 1 10 0 4 0.50% 150 0.751 1 1 1 0 0 0 5 0.50% 150 0.75 1 1 1 1 1 1 0 6 0.50% 150 0.75 1 1 1 1 110 0 7 0.50% 150 0.75 1 1 1 1 1 1 0 8 0.50% 150 0.75 1 1 1 10 10 0 0Liposomal (LMVV) Bupivacaine Pilot Study Mouse # LipoForm# Conc. Volumemg Bup 15 hr 18 hr 21 hr 1 3 2% 300 6 1 1 10 2 3 2% 300 6 1 1 0 1 4 1%450 4.5 1 1 0 2 4 1% 450 4.5 1 1 0 1 5 2% 300 6 1 1 0 2 5 2% 300 6 0 1 0

As indicated above, the numerical score to the spreadsheet wasintroduced for the evaluation of the analgesic effect of variousliposome preparations performance in vivo: For each time period (e.g. 4hrs, 8 hrs etc) a numeric value of 1 was given if the anesthesia wascomplete; 10 or 0.5 was given when analgesia was partial (incomplete)and 0 for no anesthesia. The mean for each subgroup was calculatedseparately (i.e. 1% 300 μl, 2% 150 μg).

The results show that formulation 4, where BUP was actively loaded intoLMVV with CA gradient and the iso-osmotic aqueous medium was salineprovided the best analgesic effect, although the differences between thevarious formulations was not significant, when compared to the 10 foldincrease in analgesia when compared to BUP formulations as the referenceliposomal GMV formulation [Grant et al. 2004, ibid., Bolotin et al.2000, ibid. and U.S. Pat. No. 6,162,462].

In a separate experiment the effect of repeated injection of bupivacaineloaded LMVV In mice was evaluated. The results showed (data not shown)that the analgesia obtained after the second (repeated) injection wasidentical to the one achieved at the first injection without anyobserved side effect. The conclusion was that analgesia can be prolongedby repeated injections and the time period of analgesia after the secondinjection was at least of the same duration as that obtained after thefirst injection.

1. A liposomal system comprising an aqueous medium having dispersedtherein liposomes encapsulating in their intraliposomal aqueouscompartment at least one active agent, the aqueous medium being iniso-osmotic equilibrium with said intraliposomal aqueous compartment,the liposomes having a membrane comprising liposome forming lipids, atleast one of which being sphingomyelin (SPM), the liposomal systemhaving increased stability as compared to the same liposomes free ofSPM.
 2. The liposomal system as claimed in claim 1, being stable duringlong-term storage, said stability being characterized in that no morethan 30% of the at least one active agent is present in the aqueousmedium after said storage.
 3. The liposomal system as claimed in claim2, wherein no more than 10% of the at least one active agent is presentin the aqueous medium after said storage.
 4. The liposomal system asclaimed in claim 1, wherein said SPM is a C12-C24 SPM, and is selectedfrom a synthetic or semi-synthetic SPM. 5-6. (canceled)
 7. The liposomalsystem as claimed claim 1, wherein said membrane comprises SPM in anamount between 25 to 75 mole % of the total phospholipids in saidmembrane.
 8. The liposomal system as claimed in claim 1, comprising amole ratio between the liposome forming lipids other than SPM and saidSPM in the range of 1:1 to 2:1.
 9. The liposomal system as claimed inclaim 1, wherein said liposome forming lipids have together a solidordered to liquid disordered phase transition temperature (T_(m)) above37° C.
 10. The liposomal system as claimed in claim 1, wherein saidmembrane comprises a sterol.
 11. (canceled)
 12. The liposomal system asclaimed in claim 1, wherein said liposomes are multilamellar vesicles(MLVs) or multivesicular vesicles (MVVs).
 13. (canceled)
 14. Theliposomal system as claimed in claim 1, wherein said aqueous medium andsaid intraliposomal aqueous compartment have an osmolarity between 50 to600 mOsm/kg.
 15. (canceled)
 16. The liposomal system as claimed in claim1, wherein said aqueous medium and said intraliposomal aqueouscompartment have an osmolarity difference of no more than 50 mOsmole.17-22. (canceled)
 23. The liposomal system as claimed in claim 1,wherein the mole ratio between said active agent and said liposomeforming lipids being above 0.5 mole/mole.
 24. (canceled)
 25. A methodfor storage of liposomes encapsulating in their intraliposomal aqueouscompartment at least one active agent, the liposomes having a membranecomprising liposome forming lipids, at least one liposome forming lipidbeing sphingomyelin (SPM), the method comprising forming a liposomalsystem where said liposomes are dispersed in an aqueous medium being inan iso-osmotic equilibrium with the intraliposomal aqueous compartmentof said liposomes and storing said liposomal system, said liposomalsystem having increased stability as compared to the same liposomes freeof SPM.
 26. The method as claimed in claim 25, where no more than 30% ofthe at least one active agent is present in the aqueous medium aftersaid storage. 27-33. (canceled)
 34. The method as claimed in claim 25,wherein said membrane comprises SPM in an amount between 25 to 75 mole %of the total lipids in said membrane.
 35. The method as claimed in claim25, comprising a mole ratio between the liposome forming lipids otherthan SPM and said SPM in the range of 1:1 to 2:1.
 36. The method asclaimed in claim 25, wherein said liposome forming lipids have togethera solid ordered to liquid disordered phase transition temperature(T_(m)) above 37° C. 37-42. (canceled)
 43. The method as claimed inclaim 25, wherein said aqueous medium and said intraliposomal aqueouscompartment have an osmolarity difference of no more than 50 mOsmole.44-53. (canceled)
 54. A method of treating or diagnosing of a medicalcondition comprising administering to a subject an amount of theliposomal system as claimed in claim 1.